A plasmid vector for cloning directly at the transcription initiation site of a bacteriophage T7 promoter.

The polylinker sequences in available plasmid vectors containing the bacteriophage T7 promoter are located several base-pairs from the transcription initiation site (TIS). For some applications eg. in vitro synthesis of biologically active viral RNAs (1,2), it is necessary to produce transcripts which have a minimal number of extra 5 nucleotides. Plasmid vectors which contain the SP6 promoter with either StuI (2) or BamHI (3) sites at the TIS have been described. I have now constructed a multifunctional plasmid, pT7E19(+), which has an SstI site at the TIS of the T7 promoter. The 2.8 kb plasmid is a pUC19 derivative which has the bacteriophage fl replication origin from pTZ18U (United States Biochemical) , and has a T7 promoter inserted into the polylinker. The oligo insertion destroyed the EcoRI site, but maintains the lacZ reading-frame, allowing colour screening of transformants with XGal. The figure shows the sequence of the ssDNA encapsidated after superinfection with M13K07 (4), the TIS and direction of transcription are indicated by the angled arrow. DNA fragments can be cloned at the TIS by removing the SstI 3'ends with T4 DNA polymerase (1 extra 5 G transcribed) , or by over-digestion with mung bean nuclease (3) (no extra 5 nucleotides). Dideoxy sequencing from universal primers proceeds in the direction of in vitro transcription, facilitating analysis of the junction between the promoter and a cloned fragment.